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Delft University of Technology Serpentinimonas gen. Nov., serpentinimonas raichei sp. nov., serpentinimonas barnesii sp. nov. and serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs Bird, Lina J.; Kuenen, J. Gijs; Osburn, Magdalena R.; Tomioka, Naotaka; Ishii, Shun’Ichi; Barr, Casey; Nealson, Kenneth H.; Suzuki, Shino DOI 10.1099/ijsem.0.004945 Publication date 2021 Document Version Final published version Published in International Journal of Systematic and Evolutionary Microbiology Citation (APA) Bird, L. J., Kuenen, J. G., Osburn, M. R., Tomioka, N., Ishii, SI., Barr, C., Nealson, K. H., & Suzuki, S. (2021). Serpentinimonas gen. Nov., serpentinimonas raichei sp. nov., serpentinimonas barnesii sp. nov. and serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs. International Journal of Systematic and Evolutionary Microbiology, 71(8), [004945]. https://doi.org/10.1099/ijsem.0.004945 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above. Copyright Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. TAXONOMIC DESCRIPTION Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945 DOI 10.1099/ijsem.0.004945 OPEN ACCESS Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov., Serpentinimonas barnesii sp. nov. and Serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs Lina J. Bird1,2, J. Gijs Kuenen2,3, Magdalena R. Osburn4, Naotaka Tomioka5, Shun’ichi Ishii5,6, Casey Barr2, Kenneth H. Nealson2 and Shino Suzuki5,6,7,* Abstract Three highly alkaliphilic bacterial strains designated as A1T, H1T and B1T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative and rod-shaped. Strains A1T, H1T and B1T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1T, H1T and B1T was 2 574 013, 2 475 906 and 2 623 236 bp, and the G+C content was 66.0, 66.2 and 66.1 mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera Malikia (95.1–96.4 %), Macromonas (93.0–93.6 %) and Hydrogenophaga (93.0–96.6 %) in the family Comamonadaceae. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1T, H1T and B1T against the genomes of related members in this family were below 67 %, which is below the suggested threshold for genera boundaries. Average nucleotide identity by blast values and digital DNA– DNA hybridization among the three strains were below 92.0 and 46.6 % respectively, which are below the suggested thresh- olds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov. (type strain A1T=NBRC 111848T=DSM 103917T), Serpentinimonas barnesii sp. nov. (type strain H1T= NBRC 111849T=DSM 103920T) and Serpentinimonas maccroryi sp. nov. (type strain B1T=NBRC 111850T=DSM 103919T) belonging to the family Comamonadaceae. We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs. The family Comamonadaceae, which belongs to the class hydrogen oxidizers [11], and phosphate- accumulating and Betaproteobacteria, was first described by Willems et al. [1] -removing bacteria [6, 12], and cyclohexane-degrading bacte- and now contains at least 50 genera. Most members of this ria [13]. In this study, we characterize three hyper- alkaliphilic family were isolated from soil, freshwater, activated sludge, strains (A1T, H1T and B1T) isolated from an active terrestrial hot springs and pond water [2–8]. These genera harbour a serpentinization site that represents an unusual microbial phenotypic diversity that includes aerobic organotrophs [2], habitat, as the fluids are highly alkaline, enriched in calcium, anaerobic denitrifiers [5, 9], Fe3+- reducing bacteria [10], low in sodium and have abundant dissolved hydrogen gas Author affiliations: 1Center for Bio/Molecular Science and Engineering Naval Research Lab, 4555 Overlook Ave S.W., Washington DC 20375, USA; 2Department of Earth Sciences, University of Southern California, 35 W. 37th St. SHS 560, Los Angeles, California 90089, USA; 3Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, Netherlands; 4Department of Earth and Planetary Sciences, Weinberg College of Arts & Sciences. Northwestern University Evanston, Evanston, USA; 5Kochi Institute for Core Sample Research, Japan Agency for Marine- Earth Science and Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan; 6Institute for Extra- cutting- edge Science and Technology Avant- garde Research (X- star), JAMSTEC, Natsushima 2-15, Yokosuka, Kanagawa 237-0061, Japan; 7Institute of Space and Astronautical Science (ISAS), Japan Aerospace Exploration Agency (JAXA), 3-1-1 Yoshinodai, Chuo-ku, Sagamihara, Kanagawa 252-5210, Japan. *Correspondence: Shino Suzuki, suzuki. shino2@ jaxa. jp Keywords: alkaliphile; autotrophic growth; Serpentinization; Serpentinomonas/Serpentinimonas. Abbreviations: AAI, average amino acid identity; ANIb, average nucleotide identity by blast; BS, Barnes Spring; CSM, Cedars standard medium; dDDH, digital DNA–DNA hybridization. The GenBank accession number for the 16S rRNA gene sequence of strain A1T is MT882023, for H1T it is MW414531 and for B1T it is MW411452. The genome sequence accession number of A1T is AP014568.1, for H1T it is BAWN00000000.1 and for B1T it is AP014569.1 for the chromosome and AP014570.1 for a plasmid. 004945 © 2021 The Authors This is an open- access article distributed under the terms of the Creative Commons Attribution License. 1 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945 [14–16]. Given that relatives of strains A1T, H1T and B1T neighbour- joining. Phylogenetic analysis based on 16S rRNA were dominant in various terrestrial active serpentinizing genes revealed that strains A1T, H1T and B1T belong to the sites, these strains are likely relevant to the geochemistry of family Comamonadaceae in the order Burkholderiales. The terrestrial serpentinization sites. We expect future studies on three isolates formed a distinct branch separate from other the ecology, physiology, biochemistry and molecular genetics members of Comamonadaceae with the bootstrap value of of these organisms to contribute to a better understanding of 100 (Fig. 1). The closest relatives of the three isolates were life under these extremely alkaline conditions. the genera Macromonas [4], Malikia [6] and Hydrogenophaga [11]. The samples were collected from Barnes Spring 1 (BS1) and Barnes Spring 5 (BS5; elevation 282 m, N: 38° 37.282′, W: The genome sizes of strains A1T, H1T and B1T were 2 574 013, 123° 07.987′) located at The Cedars serpentinization site in 2 475 906 and 2 623 236 bp, and the G+C contents were 66.0, northern California as described previously [17, 18]. Briefly, 66.2 and 66.1 mol%, respectively. CheckM analysis showed samples of pre- autoclaved glass beads (0.11 mm diameter that the genome completeness of the strains was over 99 % ballotini beads) that were incubated in situ in the BS1 pool at [22]. Thirty conserved marker genes were extracted from the Eh of around −250 mV for 1 week and then collected for the 14 genomes in the family Comamonadaceae and Burkholderia isolation of strains A1T and B1T. Strain H1T was isolated from cepacia in the family Burkholderiaceae, and a concatenated a sample of pool BS5 water. The samples were inoculated in alignment (5823–6689 amino acids) was generated in the sterile Cedars standard medium (CSM) 1 containing 0.05 mm CheckM platform [23]. A maximum-likelihood phylog- Na2SO4, 0.378 mm NH4Cl, 0.05 mm MgCl2, 0.06 mm K2HPO4, enomic tree based on the concatenated alignment was gener- 10 mm CABS or Na2CO3/NaHCO3, 2 mm CaCO3 (as suspen- ated by using mega X [24] and the JTT matrix-based model sion), 4 mm sodium acetate, 10 ml l−1 of ATCC trace mineral with 100 resamples. This phylogenomic tree indicated that supplement and 10 ml l−1 of ATCC vitamin solution. The pH strains A1T, H1T and B1T are deeply branching among those was adjusted to pH 10.5–11.2 using NaOH. Gas phase of the of closely related genera, as seen in the tree based on 16S media in stoppered serum vials was replaced with a mixture rRNA genes (Fig. 2). of O /H /N /Ar (2.6 : 50 : 9.8 : 37.6 by volume at 1 atm). After 2 2 2 Amino acid identity (AAI) values
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  • High-Rate Aerobic Treatment Combined with Anaerobic Digestion and Anammox

    High-Rate Aerobic Treatment Combined with Anaerobic Digestion and Anammox

    High-Rate Aerobic Treatment Combined With Anaerobic Digestion and Anammox Project code: 2013/4006 Prepared by: Huoqing Ge, Damien Batstone, Jurg Keller Date Published: June 2015 Published by: Australian Meat Processor Corporation AMPC acknowledges the matching funds provided by the Australian Government to support the research and development detailed in this publication. Disclaimer: The information contained within this publication has been prepared by a third party commissioned by Australian Meat Processor Corporation Ltd (AMPC). It does not necessarily reflect the opinion or position of AMPC. Care is taken to ensure the accuracy of the information contained in this publication. However, AMPC cannot accept responsibility for the accuracy or completeness of the information or opinions contained in this publication, nor does it endorse or adopt the information contained in this report. No part of this work may be reproduced, copied, published, communicated or adapted in any form or by any means (electronic or otherwise) without the express written permission of Australian Meat Processor Corporation Ltd. All rights are expressly reserved. Requests for further authorisation should be directed to the Chief Executive Officer, AMPC, Suite 1, Level 5, 110 Walker Street Sydney NSW. Executive Summary Australian red meat processing facilities can produce significant volumes of wastewater during slaughtering and cleaning operations. This wastewater stream is typically characterised by highly variable levels of suspended solids, organic matter and nutrient compounds, sometimes at concentrations more than four times greater than domestic sewage. It is important to treat this wastewater before discharging it into the environment or sewers. Typical treatment methods involve pre-treatment (dissolved-air flotation), followed by treatment in anaerobic lagoons to remove organic matter, and removal of biological nutrients – often by adding chemicals to remove phosphorus (P) if required.